The present invention is concerned with sequence analysis of saccharide material and it is especially applicable to the sequencing of saccharide chains containing numerous amino sugar residues such as, for example, are found in glycosaminoglycans (GAG""s) which include the biologically important polysaccharides, heparan sulphate (HS) and heparin.
Heparan sulphate (HS) and heparin are chemically-related linear glycosaminoglycans (GAG""s) composed of alternate xcex1,xcex2-linked glucosamine and hexuronate residues with considerable structural variation arising from substitution with acetyl and N- and O-sulphate groups, and from the presence of D- and L-isomers of the hexuronate moieties. These polysaccharides are of fundamental importance for many diverse cellular and biochemical activities. Their regulatory properties are dependent on their ability to bind, and in some cases to activate, protein molecules which control cell growth, cell adhesion, and enzyme-mediated processes such as haemostasis and lipid metabolism. However, analysis of protein-binding monosaccharide sequences in HS/heparin is generally difficult and a universal procedure suitable for routine use has not been described to date.
An object of the present invention is to provide a new method of sequence analysis of saccharide fragments such as oligosaccharides that may be derived from HS (or heparan sulphate proteoglycan HSPG) and heparin, this method enabling rapid elucidation of recognition sites and other sequences of interest and thereby facilitating the rational design of synthetic compounds to serve as drugs for therapeutic modulation of polysaccharide function.
In one aspect the invention may be regarded as being based on a concept of bringing about a preliminary partial depolymerisation by scission of specific intrachain linkages in reducing end referenced saccharide chains, such as for example HS/heparin saccharide chains, followed by exoenzyme removal of non-reducing end (NRE) sugars or their sulphate groups so as to yield a range of labelled fragments that can be separated by gel electrophoresis or other appropriate techniques to give a read-out of the sequence of sugar units and their substituents. Although the invention may be described mainly in relation to saccharides that are found in heparan sulphate and heparin, the basic principle of the sequencing strategy is applicable to many other GAG""s and different saccharides, including the saccharide component of glycoproteins.
Use of exoenzymes, in particular exoglycosidsases, for removal of terminal sugar residues at the non-reducing end of saccharide chains has previously been proposed in connection with methods for sequencing such chains, for instance in WO 92/02816 and in WO 92/19974 and WO 92/19768. However, in these prior art proposals there has been no preliminary step of partial depolymerisation of the saccharide material, involving cleavage of internal glycosidic linkages, before treatment with said exoenzymes. In WO 92/02816 for example, it is proposed in relation to a saccharide sequencing method disclosed therein to use exoenzymes successively to remove and identify terminal sugar residues at the non-reducing end of initially undegraded saccharide chains, and to carry out a series of sequential steps with the residual saccharide material being recovered after each step before proceeding to the next. In WO 92/19974 and WO 92/19768, although exoenzymes are mentioned inter alia as possible sequencing agents, again it is proposed that these be applied sequentially direct to an oligosaccharide being analysed in an iterative process without a preliminary partial depolymerisation step as required by the present invention. In all these prior art methods the sequencing information is obtained and presented in a different manner to that in the present invention.
An acknowledgement is also made of a paper by Kyung-Bok Lee et al, Carbohydrate Research, 214 (1991), 155-168, which refers to the use of exoglycosidases and of endoglycosidases in connection with sequencing of oligosaccharides. This publication does not, however, disclose the combined use of both exoglycosidases and endoglycosidases in sequence in the same manner as herein defined in the claims relating to the present invention.
More specifically, the present invention broadly provides a method of analysing and sequencing saccharide material comprising saccharide chains which contain more than three monosaccharide units interconnected through glycosidic linkages that are not all identical and which each include a referencing feature at their reducing end, wherein selected exoenzymes comprising exoglycosidases of known specificity that cleave only particular glycosidic linkages at the non-reducing end of saccharide chains are used to obtain sequence information, said method being characterised in that it comprises the sequential steps of:
(a) subjecting said saccharide material to partial depolymerisation by controlled treatment with a selective scission reagent that acts in accordance with a known predetermined linkage specificity as an endoglycosidase to cleave a proportion of susceptible internal glycosidic linkages, that is, susceptible glycosidic linkages spaced from the non-reducing end of the saccharide chains, thereby to produce a mixed set of saccharide chains, intact chains and fragments of intact chains, having different lengths representative of the full spectrum of all possible lengths given the particular glycosidic linkage specificity of the selective scission reagent employed,
(b) treating a selected sample or samples of said mixed set of saccharide chains and chain fragments with said exoenzymes, either singly or in combination in accordance with a predetermined strategy, to an extent sufficient to obtain complete digestion and cleave susceptible linkages at the non-reducing end of all the saccharide chains, and then
(c) analysing said sample or samples to detect the various saccharide chain fragments generated by the cleavage treatments which are present therein and which have a reducing end derived from the reducing end of the corresponding chain in the original saccharide material, and obtaining, collectively from the results of said analysis, information enabling the monosaccharide sequence in the original saccharide material to be at least partially deduced.
In carrying out this saccharide sequencing method of the present invention, the saccharide material will generally be treated, usually before the controlled partial depolymerisation step, to modify the saccharide chains at their reducing ends in order to introduce the reducing end referencing feature for providing a common reference point or reading frame to which the monosaccharide sequence can be related and for facilitating, during analysis, detection of chain fragments having a reducing end derived from the reducing end of the corresponding chains in the original saccharide material. This end referencing feature is conveniently provided by selectively labelling or tagging the monosaccharide units at the reducing ends of the saccharide chains, using for example radiochemical, fluorescent, biotin or other calorimetrically detectable labelling means.
If low pH nitrous acid is used for carrying out the partial depolymerisation of the saccharide material as hereinafter described, a presently preferred fluorescent labelling agent is anthranilic acid as referred to in more detail later. However, if a selective scission reagent other than nitrous acid is used for bringing about the partial depolymerisation, e.g. an endoglycosidase enzyme, an aminocoumarin hydrazide, e.g. 7-amino4-methylcoumarin-3-acetyl hydrazide, may be preferred for providing a fluorescent labelling agent having a relatively high labelling efficiency. For use as a radiochemical labelling agent tritiated borohydride may be used.
In an alternative but usually less preferred technique for providing end-referenced saccharide chains or chain fragments, the chains may be immobilized by coupling the reducing ends to a solid phase support. This can then permit those chain fragments, produced by the partial depolymerisation treatment, which are not contiguous with the reducing ends of the original undegraded chains to be physically separated and removed, whereupon subsequent release of the immobilized chain fragments from the solid phase support then provides the required mixed set of chain fragments ready for exoenzyme treatment as before.
In preferred embodiments, as hereinafter more fully described, electrophoretic separation means such as polyacryiamide gel electrophoresis (PAGE), e.g. gradient PAGE, will usually be used for detecting the fragments produced by the cleavage treatments, these fragments being separated according to differences in length and composition which are reflected in different mobilities in the electrophoretic medium. If necessary, for uncharged or lightly charged saccharide chains, the material can be treated in a preliminary operation so as to incorporate therein suitable electrically charged groups in a known manner in order to permit the use of electrophoretic separation techniques. This will not usually be necessary, however, in sequencing HS or heparin oligosaccharides which already contain a significant number of charged sulphate and carboxyl groups. Other alternative separation techniques, for example capillary electrophoresis or high performance liquid chromatography (HPLC), may also be used for detecting the fragments so long as the requisite resolving power is available.
After the controlled partial depolymerisation step the mixed set of saccharide chain fragments produced will usually be used to provide a number of separate samples. One of these samples, and generally a control sample of the original material, will then be subjected to the separation technique, e.g. gradient PAGE, to separate and detect the different fragments present for reference purposes before exoenzyme treatment. At the same time, other samples of the set of fragments will also be subjected to the same separation technique so as to separate and detect the different saccharide fragments present after each of these other samples has been treated with a different exoenzyme or combination of exoenzymes.
In applying the invention to the sequencing of saccharide chains containing many amino sugar residues, such as are found in glycosaminoglycans (GAG""s) for which the method is especially useful, the preliminary controlled partial depolymerisation involving cleavage of specific internal glycosidic linkages is most conveniently carried out as hereinafter more fully described using nitrous acid at low pH as a chemical selective scission reagent. It is also possible, however, in some cases as an alternative to a chemical selective scission agent to use appropriate enzymatic endoglycosidases, e.g. the bacterial lyases heparinase (EC 4.2.2.7) or heparitinase (EC 4.2.2.8), under suitable conditions to bring about selective enzymatic cleavage of internal glycosidic linkages.
As GAG""s and similar saccharides also generally contain various sulphated monosaccharide units, the selected exoenzymes used for treating tile fragments obtained after the initial hydrolysis and partial depolymerisation will usually include, in addition to exoglycosidases, selected exosulphatases for effecting a controlled removal of particular sulphated groups from specific terminal monosaccharide residues at the non-reducing end of the chains. Other additional specific enzymes may also be used in analysing the fragments obtained after the partial depolymerisation as part of the overall strategy selected for extracting or confirming the sequence information required.
Examples of selective scission reagents which may be used in carrying out the sequencing method of the present invention include the following:
The enzymes mentioned above are exoenzymes which act specifically to remove the terminal sugar residues or their sulphate substituents at the non-reducing end (NRE) of glycan fragments. Details of many such enzymes are readily available in the literature, and by way of example reference may be had to an informative review article entitled xe2x80x9cEnzymes that degrade heparin and heparan sulphatexe2x80x9d by John J. Hopwood in xe2x80x9cHeparin: Chemical and Biological Properties, Clinical Applicationsxe2x80x9d, pages 191 to 227, edited by D. A. Lane et al and published by Edward Arnold, London, 1989, and to another review article entitled xe2x80x9cLysosomal Degradation of Heparin and Heparan Sulphatexe2x80x9d by Craig Freeman and John Hopwood in xe2x80x9cHeparin and Related Polysaccharidesxe2x80x9d, pages 121 to 134, also edited by D. A. Lane et al and published by Plenum Press, New York, 1992.
Some of these enzymes are available commercially and others can be isolated and purified from natural sources as described in the literature. Moreover, in some cases recombinant versions are known and, when available, these will often be preferred because of a high level of purity that can usually be achieved. Published papers in which the isolation and preparation or properties of some of the enzymes referred to above are described include: Alfred Linker, (1979), xe2x80x9cStructure of Heparan Sulphate Oligosaccharides and their Degradation by Exo-enzymesxe2x80x9d, Biochem. J., 183, 711-720; Craig Freeman and John J Hopwood, (1992), xe2x80x9cHuman xcex1-L-iduronidasexe2x80x9d, Biochem. J., 282, 899-908; Wolfgant Rohrborn and Kurt Von Figura, (1978), xe2x80x9cHuman Placenta xcex1-N-Acetylglucosaminidase: Purification. Characterisation and Demonstration of Multiple Recognition Formsxe2x80x9d, Hoppe-Seyler""s Z. Physiol. Chem., 359, 1353-1362; Craig Freeman and John J Hopwood, (1986), xe2x80x9cHuman Liver Sulphamate sulphohydrolasexe2x80x9d, Biochem. J., 234, 83-92: Craig Freeman, et al, (1987), xe2x80x9cHuman Liver N-acetylglucosamine-6-sulphate sulphatasexe2x80x9d, Biochem. J., 246, 347-354; Craig Freeman and John J Hopwood, (1991), xe2x80x9cGlucuronate-2-sulphatase activity in cultured human skin fibroblast homogenatesxe2x80x9d. Biochem. J,. 279. 399-405: Craig Freeman and John J Hopwood, (1987), xe2x80x9cHuman liver N-acetylglucosamine-6-sulphate sulphatasexe2x80x9d, Biochem. J., 246. 355-365: Irwin G. Leder (1980), xe2x80x9cA novel 3-O sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-xcex1-D-glucopyranoside 3-sulphatexe2x80x9d, Biochemical and Biophysical Research Communications, 94, 1183-1189; Julie Bielicki, et al, (1990), xe2x80x9cHuman liver iduronate-2-sulphatasexe2x80x9d, Biochem. J., 271. 75-86: Irwin G. Leder (1980), xe2x80x9cA novel 3-O sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-xcex1-D-glucopyranoside 3-sulphatexe2x80x9d, Biochemical and Biophysical Research Communications, 94, 1183-1189; and Julie Bielicki, et al, (1980), xe2x80x9cEvidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparinxe2x80x9d, Biochemistry, 77, 6551-6555.
A recombinant version of an exoenzyme and the preparation thereof is described for example in connection with a synthetic xcex1-L-iduronidase in international patent publication WO 93/10244.
The contents of the above-mentioned publications are incorporated herein by reference.
The nitrous acid (HNO2) reagent used at low pH cleaves hexosaminidic linkages when the amino sugar is N-sulphated (GlcNSO3) irrespective of the position of the linkage in the saccharide chain, but most importantly GlcNAcxe2x86x92GlcA linkages are resistant to HNO2 scission. The controlled hydrolysis and partial depolymerisation with nitrous acid can be achieved by preparing the reagent as described by Steven Radoff and Isidore Danisliefsky, J. Biol. Chem. (1984), 259, pages 166-172 a publication of which the content is also incorporated herein by reference. A typical example with practical details, however, is described below.
The saccharide to be treated (1-2 nmoles) is dried down by centrifugal evaporation, dissolved in 80 xcexcL of distilled H2O and cooled on ice. To this solution is added 10 xcexcL of 190 mM HCl and 10 xcexcL of 10 mM NaNO2, both precooled on ice. These reactants are mixed by vortexing and incubated on ice. At predetermined time points (for example 0, 20, 40, 60, 90 and 120 minutes), aliquots of the reaction mixture are removed and the low pH HNO2 hydrolysis is stopped either by addition of excess ammonium sulphamate to quench the reagent, or by raising the pH above 4.0 (for example by addition of Na2CO3 solution). It has in fact been found most convenient to stop the reaction by addition of 1/4 volume of 200 mM sodium acetate buffer, pH 5.0. This raises the pH to approximately pH 4.3-4.4 and provides buffer conditions immediately compatible with subsequent enzyme treatments, thus avoiding the need for any further clean-up steps such as removal of salts or buffer exchanges. Finally, once all the time points are complete the aliquots are remixed and pooled. This is crucial since it creates a mixed set of saccharide products, hydrolysed partially and at random, which contain fragments corresponding to all possible cleavage positions, whereas a single time point would not create such a representative set. Thus, the fragments have different lengths ranging throughout the full spectrum of possible lengths for the particular glycosidic linkage specificity of the HNO2 reagent, and ideally there should be a fairly even distribution of the different length fragments.
In carrying out the invention, it will be appreciated that in effect the controlled, incomplete hydrolysis of N-sulphated disaccharides by the HNO2 treatment, i.e. the partial HNO2 scission or depolymerisation (herein denoted as pHNO2), is used to xe2x80x9copen-upxe2x80x9d the glycan structure of the saccharide material under analysis so as to expose a range of NRE sugars and sulphate groups to attack by specific exoglycosidases and exosulphatases. Indeed, this dual approach of combining a preliminary controlled hydrolysis and partial depolymerisation involving cleavage of internal linkages with a progressive action of exoenzymes acting at the non-reducing end of the fragments produced can be regarded as being an important and significant key feature characterising the sequencing method of this invention.
The invention and the manner in which it may be carried out will now be hereinafter described in more detail with reference to non-limiting illustrative examples.